Hydroxysafflor yellow A attenuates the expression of inflammatory cytokines in acute soft tissue injury

We examined the effect of hydroxysafflor yellow A (HSYA) on the inflammatory response to strike-induced acute soft tissue injury in rats. Soft tissue injury was induced in rat leg muscles using a strike hammer, followed by intraperitoneal administration of HSYA at 16, 32, or 64 mg/kg. After 24 h, the rats were anaesthetized, blood and muscle samples were taken. Plasma levels of interleukin (IL)-6, IL-1β, and tumour necrosis factor (TNF)-αwere measured by enzyme-linked immunosorbent assay. Total RNA and protein were isolated from muscle tissue to determine the mRNA levels of IL-6, IL-1β, TNF-α, vascular cell adhesion molecule (VCAM)-1, and intercellular adhesion molecule (ICAM)-1, and the protein level of phosphorylated p38 mitogen-activated protein kinase (MAPK). Nuclear factor (NF)-κB expression was determined by muscle histopathology and immunohistochemistry. HSYA attenuated pathologic changes instrike-induced soft tissue inflammation. Treatment with HSYA also alleviated strike-induced increases in TNF-α, IL-1β, IL-6, VCAM-1, and ICAM-1mRNA levels and inhibited the increased activation of NF-κB and phosphorylation of p38 MAPK in muscle tissue. These findings suggest that HSYA effectively inhibits strike-induced inflammatory signal transduction in rats.

Effects of HSYA on plasma IL-6, IL-1β, and TNF-α protein concentrations in rats. Plasma IL-6, IL-1β , and TNF-α protein levels were significantly increased in the strike group compared with the sham group, but were attenuated in the strike+ HSYA and strike+ DXM groups (Fig. 4).
Effects of HSYA on p38 mitogen-activated protein kinase (MAPK) activation. p38MAPK is a known intracellular signal transducer of the inflammatory reaction, which can be triggered by various extracellular stimuli. p38 MAPK activation in muscle tissue after striking and HSYA treatment were assessed by western blot analysis. The level ofphosphorylated p38 MAPK was markedly augmented int he strike group, and this augmentation was attenuated by HSYA and DXM (Fig. 5).
Effect of HYSA onnuclear factor (NF)-κB p65. We assessed the expression level of NF-κ B subunit p65 by immunohistochemistry to determine if the observed anti-inflammatory effect of HSYA involved inhibition of NF-κ B activity. The proportion of p65-positive cells was significantly increased in the strike group compared with the sham group, and treatment with HSYA or DXM significantly attenuated the strike-induced increase in p65-positive cells (Fig. 6).

Discussion
Acute soft tissue injury is a common orthopaedic condition 6 . It is characterised by a series of acute contusions and/or tears in tissues beneath the skin, including muscle, ligament, fascia, tendon, synovium, fat, joint capsule peripheral nerves, and blood vessels, but excluding bones. Acute soft tissue injuries are generally induced by external stress above a threshold, causing symptoms such as localized swelling, pain, dysfunction, and bruising. The main pathological change in acute soft tissue injury involves traumatic aseptic inflammation, characterized by local tissue necrosis, blood capillary dilation, leukocyte infiltration, oedema, and haemorrhage.
An acute soft tissue injury model induced by a blunt blow has recently been used widely to study the anti-inflammatory effects of numerous drugs 7,8 . Although its aetiology differs from that of clinical acute soft tissue injuries, its pathology and clinical pathological manifestations are similar 7 . In the current study, we used a self-made blunt-blow device to establish a rat model of acute soft tissue injury to simulate the occurrence of clinical acute soft tissue injury and to examine the therapeutic effect HSYA on such injuries. Corticosteroids are commonly used for treating acute and chronic soft tissue injuries in the clinic 9 , and we therefore used DXM as a positive control drug to evaluate the efficacy of HSYA for the treatment of acute soft tissue injury.
Local swelling and bruising are the main manifestations of acute soft tissue injury, and are the basis for vascular-lesion rupture or damage, oozing red blood cells, inflammatory cells, and muscle fibre breaks, degeneration, and necrosis. Using HE staining, we showed that the muscle fibre structure was destroyed and there was severe skeletal muscle tissue oedema following trauma in our rat model, while HSYA relieved the oedema and bleeding.
Blunt blow causes an aseptic inflammatory response, fibroblast proliferation, and subsequent collagen deposition. Neutrophils also play an important role in the pathogenesis of acute soft tissue injury and are released early from traumatic lesions to induce free radicals, which subsequently damage cells in local tissues. Neutrophils release numerous inflammatory mediators, including TNF-α , IL-1β , IL-6, IL-8, IL-12, vasoactive amines, and arachidonic acid metabolites, which further exacerbate the adhesion and aggregation of inflammatory cells and the release of inflammatory factors, and concurrently activate new inflammation. TNF-α and IL-1β are two of the major proinflammatory cytokines involved in the early inflammatory response, causing a variety of biochemical effects [10][11] . IL-6 promotes neutrophil activation and aggregation, and IL-6 levels partially reflect the intensity of tissue injury 12 . Increasesin TNF-α and IL-1β induce the expression of adhesion molecules such as ICAM-1, resulting inlymphocyte adhesion to the endothelial cell surface 13 . HSYA may relieve blood circulation disorders 14 , and Safflor Yellow (the main ingredient of which is HSYA) can inhibit the increased blood capillary permeability caused by inflammation damage 15 . Leukocyte activation 16 , white blood cell adhesion to epithelial cells 5 , and endothelial cell adhesion molecule expression 17 can all be inhibited by HSYA, and leukocyte infiltration was demonstrated in the current experiment. The results of the current study showed that strike-induced acute soft tissue injury in rats enhanced the mRNA and protein expression levels of TNF-α , IL-1β , IL-6, ICAM-1, and VCAM-1, and these were all significantly inhibited by HSYA treatment. In addition, the HYSA-induced decrease in inflammatory cytokine expression was associated with reduced oedema and bleeding.
Inflammatory mediators (e.g., TNF-α and IL-1β ) can activate multiple intracellular signalling molecules, such as MAPK and NF-κ B, and thus trigger the expression of related genes 18,19 . During acute soft tissue injury, skeletal muscle endothelial cells, macrophages, and neutrophils secrete a variety of proinflammatory cytokines and proteases that take part in early inflammation through the NF-κ B and MAPK pathways. In this study, the proportion of p65-positive cells in muscle was significantly increased by striking, and significantly attenuated by treatment with HSYA or DXM. We therefore hypothesized that HSYA inhibited the NF-κ B signalling pathway in skeletal muscle tissue. HSYA treatment also significantly decreased strike-induced p38 MAPK phosphorylation in skeletal  In summary, HSYA reduced strike-triggered local oedema and neutrophil infiltration in skeletal muscle in a dose-dependent manner. The mechanism of HYSA action appears to involve inhibition of p38 MAPK phosphorylation and suppression of NF-κ B pathway activation, thus decreasing the gene and protein expression levels of TNF-α , IL-1β , IL-6, ICAM-1, VCAM-1, and other inflammatory mediators.

Chemicals and reagents.
Dried safflower (C. tinctorius L.) flowers were obtained from Tacheng, Xinjiang Uygur Autonomous Region, China, and identified by Professor Jiashi Li (Beijing University of Chinese Medicine). HSYA was isolated and purified from aqueous extracts of C. tinctorius L. by macroporous resin-gel column chromatography, as described previously 20 , with a purity of 95.9% determined by high-performance liquid chromatography (HPLC) (Fig. 7). HSYA was dissolved insterile 0.9% NaCl for subsequent use. The molecular weight of HSYA was 612, and its molecular structure is displayed in Fig. 1 HPLC analysis of HSYA. HPLC analyses were performed with an Apollo C18 column (250 mm × 4.6 mm, 5 μ m; Grace Davison (Curtis Bay, MD, USA)) on an LC-10AT HPLC system with an SPD-6AV UV detector (Shimadzu, Kyoto, Japan). The mobile phase consisted of acetonitrile (A) and 0.1% trifluoroacetic acid (B) at a flow rate of 1.0 mL/min. The gradient elution program was as follows: initial 1% solvent A and 99% solvent B; from 0-50 min, solvent A was linearly increased from 1% to 35%, and solvent B was linearly decreased from 99% to 65%; from 50-60 min, solvent A was linearly increased from 35% to 45%, and solvent B was linearly decreased from 65% to 55%. The optical absorbance was monitored at 405 nm and the column temperature was 30 °C. The HSYA purity was determined quantitatively by the area normalization method. Experimental design and treatment. Eighty-four Wistar rats were divided randomly into seven groups: sham group (saline 64 mg/kg, intraperitoneal [i.p.]), HSYA blank group (HSYA 64 mg/kg, i.p.), strike group (strike + saline), strike+ HSYA groups (strike + HSYA 16, 32, or 64 mg/kg, i.p.), and strike + DXM group (strike + DXM 5 mg/kg, i.p.). All rats were anaesthetized with 1% pentobarbital i.p. The medial thigh 1 cm from the knee joint was depilated with depilatory cream. A soft tissue injury model was established using a self-made hammer as follows: a stainless steel hammer weighing 267 g, with a bottom-surface radius of 0.5 cm (Fig. 8), was dropped through the central vertical axis of a plastic tube from a height of 30 cm to hit the medial thigh, 1 cm from the knee joint. In the sham groups, a marker pen was used instead of the stainless steel hammer. Rats in the sham and strike groups were injected i.p. twice with sterile 0.9% NaCl at 30 min and 6 h after the strike. Rats in the strike+ HSYA and strike+ DXM groups were then injected i.p. with the indicated doses of HSYA or DXM, respectively. At 24 h after the strike, the rats were anaesthetized with pentobarbital sodium, blood samples were obtained from the abdominal aorta using a heparinised syringe for plasma preparation, and the rats were sacrificed by exsanguination. Injured muscle tissues were cut and washed in ice-cold saline, and muscle tissues from the right leg were snap-frozen in liquid nitrogen for RNA isolation and protein extraction. Muscle tissues from the left leg were trimmed and fixed in 4% paraformaldehyde solution for histopathological and immunohistochemical examinations. Immunohistochemical detection. After treatment with xylene and hydration with graded alcohols, the muscle samples were incubated in citrate buffer (pH 6.0) at 96 °C for 20 min for antigen retrieval. Samples were washed three times in phosphate-buffered saline (PBS), blocked with rabbit serum for 30 min, and then incubated with primary antibody against p65 (1:100) at 4 °C overnight. After washing with PBS, the samples were incubated in biotinylated rabbit anti-goat antibody for 60 min at 37 °C. Some sections were also incubated exclusively with primary antibody or exclusively with secondary antibody to verify the binding specificity, which confirmed no positive staining in these sections. Digital images were captured at 100× magnification from five randomly selected fields for each section, and positive areas were integrated using the NIS-ELEMENTS quantitative automatic program (Nikon). The average optical absorbance was regarded as the level of target protein.
Western blot analysis. Phosphorylated p38 MAPK levels in muscle tissues were determined by western blot analysis. Protein concentration was determined using the bicinchoninicacid method, and equal amounts were loaded on a 12% sodium dodecyl sulphate-polyacrylamide gel for electrophoresis. Protein bands were then transferred onto nitrocellulose membranes and blocked with Tris-buffered saline 0.1% Tween 20 (TBST) containing 5% non-fat dried milk. The membranes were incubated with primary antibodies overnight at 4 °C. Mouse monoclonal anti-p38 MAPK antibody and anti-phospho-p38 MAPK antibody were diluted 1:1000. Following incubation, the membranes were washed three times with TBST, followed by incubation with horseradish peroxidase-conjugated goat anti-rabbit antibody (diluted 1:1000 in TBS). Reactive proteins were detected using a chemiluminescent solution, and the bands were quantified using an Odyssey Infrared Imaging System (Lincoln, NE. US). The internal control for phospho-p38 MAPK was p38 MAPK.
ELISA. Blood samples were obtained from the abdominal aorta using a heparinised syringe, and centrifuged at 1100 × g for 10 min to prepare plasma. TNF-α , IL-1β , and IL-6 concentrations in the plasma were measured by ELISA, according to the manufacturer's protocols.
Statistical analysis. All data are expressed as mean ± SD. Statistical analyses were performed using one-way analysis of variance with two-tailed tests and Student-Newman-Keuls multiple comparison tests, using SPSS 19.0 software. Figures were generated using GraphPad Prism 5.0 software. A value of p < 0.05 indicated a significant difference.